It is known that, in living bodies and life phenomena, free radical species such as nitrogen monoxide act as second messengers for signal transduction, as well as control blood pressure in the circulatory system. It has also been shown that superoxides and hydrogen peroxide play important roles in the proper functioning of the immune system and the like. However, while singlet oxygen (1O2) has an analogous electronic structure to these reactive oxygen species, its physiological role is not as well delineated.
Early work on the physiological role of singlet oxygen by Howes and Steele, Res. Commun. Chem. Pathol. Pharmacol. 2:619-626 (1971); Res. Commun. Chem. Pathol. Pharmacol. 3:349-357 (1972) suggest its possible involvement in liver microsomal hydroxylation reactions. Today, singlet oxygen is recognized as the principal bacterial oxidizing agent employed by human neutrophil (macrophage) and monocyte phagosome. In addition, singlet oxygen has been revealed to be a reactive species of photodynamic therapy, a form of cancer treatment. Roles for singlet oxygen in signal transduction as well as in oxidase and peroxidase-catalyzed reactions have also been theorized. A better understanding of the activity of singlet oxygen in living systems is therefore of great importance.
Numerous methods are known for the measurement of singlet oxygen in living systems. These methods employ chemiluminescence, luminescence, and electron spin resonance (ESR), among other tools. Unfortunately, current detection techniques are unreliable, as they all possess low specificity and sensitivity for singlet oxygen. In the past, fluorescent materials have provided the specificity and sensitivity needed to image other reactive oxygen species. Thus, an oxidatively sensitive fluorogenic probe that becomes stable and intensely fluorescent upon oxidation, emits fluorescence within a comparatively narrow wavelength range and can be used in conjunction with other dyes without interfering with their signals would represent a significant advance in the art. Furthermore, probes that localize within cellular compartments, e.g., cytosol, mitochondria, etc., particularly those implicated in the formation or reaction of singlet oxygen, would allow researchers to probe singlet oxygen formation and distribution at the subcellular level. The present invention provides such fluorogenic probes, conjugates including the probes and methods of using both the probes and the conjugates.